To evaluate the validity and acceptability of at-home self-collection to test for high-risk human papillomavirus (HPV) and sexually transmitted infections among women overdue for cervical cancer screening by national guidelines. Low-income, infrequently screened women were recruited from the general population in North Carolina to participate in an observational study. Participants provided two self-collected cervicovaginal samples (one at home and one in the clinic) and a clinician-collected cervical sample. Samples were tested for high-risk HPV, Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, and Mycoplasma genitalium. Cervical samples were also tested by liquid-based cytology. Overall, 193 women had conclusive high-risk HPV results for all three samples and cytology results. Prevalence of high-risk HPV within self-home samples (12.4%) was not different from that within clinician samples (11.4%; P=.79) and from that within self clinic samples (15.5%; P=.21). Positivity for high-risk HPV in all sample types increased with increasing grades of cervical abnormality (P<.001). Self-home samples detected high-risk HPV in all identified cases of high-grade squamous intraepithelial lesions and of cervical intraepithelial neoplasia 2 or worse. Detection was comparable across sample types for T vaginalis (range 10.2-10.8%), M genitalium (3.3-5.5%), C trachomatis (1.1-2.1%), and N gonorrhoeae (0-0.5%). Kappa values between sample types ranged from 0.56 to 0.66 for high-risk HPV, 0.86-0.91 for T vaginalis, and 0.65-0.83 for M genitalium. Most participants reported no difficulty understanding self-collection instructions (93.6%) and were willing to use self-collection in the future (96.3%). Mail-based, at-home self-collection for high-risk HPV and sexually transmitted infection detection was valid and well accepted among infrequently screened women in our study. These findings support the future use of high-risk HPV self-collection to increase cervical cancer screening rates among higher risk women in the United States.